The Single-Nucleotide Polymorphism (SNP) Validity to Detect Omicron Variants

Introduction Mutation of SARS-CoV-2 has generated several variants of concern (VOC) which spread promptly worldwide. These emerging variants affected global strategies to overcome COVID-19. Variants of SARS-CoV-2 are determined by the whole genome sequencing (WGS) assay, which is time-consuming, with limited availability (only in several laboratories). Hence, a faster and more accessible examination is needed. The single-nucleotide polymorphism (SNP) method is one of the options for genomic variation surveillance that can help provide an answer to this challenge. This study aims to determine the validity of the SNP method with PCR to detect omicron variants of SARS-CoV-2 compared with the gold standard, WGS. Methods This is a diagnostic analysis of 140 confirmed COVID-19 nasopharyngeal samples taken from the Kemayoran COVID Emergency Hospital Laboratory and the West Java Provincial Health Laboratory from April to October 2022. Data analysis was carried out to determine conformity and validity values. Results Analysis using Cohen's kappa coefficient test showed high conformity between SNP and WGS (p value <0.001; kappa coefficient = 0.948). SNP showed great validity values on omicron BA.1 (90% sensitivity; 100% specificity), omicron BA.2 (100% sensitivity; 99% specificity), and omicron BA.4/5 (99.2% sensitivity; 100% specificity). Conclusion The SNP method can be a more time-efficient alternative to detect omicron variants of SARS-CoV-2 and distinguish their sublineages (BA.1, BA.2, and BA.4/5) by two different specific gene mutations in combination analysis (ΔH69/V70 and Q493R mutations).


Introduction
Te outbreak of a novel coronavirus infection originating from the Hubei province of China caused a worldwide pandemic at the end of 2019 [1].On February 2020, the World Health Organization (WHO) named this novel coronavirus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and named the diseases as coronavirus-disease of 2019 .For the past two years, various eforts have been made to overcome the COVID-19 pandemic by conducting strategies regarding examination (testing), isolation, lockdown, and intensive vaccination [2].
Since its frst emergence at the end of 2019, SARS-CoV-2 has mutated and generated several variants of concern (VOC) that have rapidly spread globally [3].On December 29, 2021, there was an increase in COVID-19 cases in the world (10,448,409 cases daily), peaked on January 24, 2022 (23,555,167 cases daily), decreased on April 18, 2022 (4,713,853 cases daily), and then peaked again on July 18, 2022 (7,172,704 cases daily) [4].Te COVID-19 cases in Indonesia experienced a signifcant increase on June 14, 2021 (78,551 cases daily), peaked on July 12, 2021 (350,273 cases daily), and then experienced a decrease in cases on October 4, 2021 (8,648 cases daily).Ten, there was a spike in cases again in February 2022 which peaked on February 14, 2022 [5].Tis surge in the number of cases has raised suspicions about a change in the pattern of infection of the SARS-CoV-2 virus variant in Indonesia.Te emergence of SARS-CoV-2 variants has afected strategies to overcome COVID-19 [6].A variant of SARS-CoV-2 is classifed as VOC when it has increased transmissibility and/or virulence, and when it has created some changes in disease presentation, diagnostic methods, and management measurement [7].Variants of SARS-CoV-2 are determined by whole genome sequencing (WGS) examination.Unfortunately, these examinations are carried out only by several laboratories in each region in Indonesia and take approximately 3-5 days.Such limitations give necessity to the need for a faster and easier examination method that can be performed in all molecular laboratories.
Te use of quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) remains the gold standard for testing, wherein unique sequences of the SARS-CoV-2 genome are detected.Te diagnostic accuracy of this technique is of utmost importance [8].Te qRT-PCR examination of single-nucleotide polymorphism (SNP) targets is one of the options for genomic variation surveillance that can help bring an answer to this issue.Tis SNP method can be carried out in all laboratories with biosafety level 2 (BSL-2) because it is PCR-based and can be performed within 2-4 hours with a much faster examination in comparison with WGS [9].Tis SNP technique can also predict COVID-19 severity with specifc vulnerable gene detection [10].
Omicron is one of the VOCs that spread globally and is regarded as one of the signifcant public health concerns [11].Omicron was frst identifed in South Africa in November 2021.Tis subvariant SARS-CoV-2 was designated as a VOC on 26 th November 2021 [12].Te diferences between omicron and the frst SARS-CoV-2 genome are as follows: the spike region of the originally BA.1 omicron genome had 35 mutations with 30 amino acid substitutions, three in-frame deletions, and an insertion of three amino acids.Fifteen mutations exist in the receptor-binding domain (RBD), which is the dominant binding site of the virus to host cells and target neutralizing antibodies (Nabs) [11,13].Omicron variants also have three and six mutations in the region coding for membrane protein and nucleocapsid protein.Spike protein is the major surface glycoprotein of SARS-CoV-2, which is divided into the Nterminal domain (NTD) and receptor-binding domain (RBD).A small group of 25 amino acids of RBD is responsible for interaction with cellular receptor angiotensinconverting enzyme-2 (ACE-2).Following ACE-2 binding, S1 is cleaved and detached, whereas S2 undergoes a major conformational change to expose the fusion loop, which mediates the fusion of viral and host membranes, allowing viral RNA to enter the host cell cytoplasm and commence the replicative cycle [14].Some of the mutations in the NTD spike region of omicron have been observed previously in other variants, for example, del69-70 (ΔH69/V70/S-gene test failure/SGTF) in the alpha variant, T951 in kappa and iota variants, and G142D in kappa and delta variants [11].
Substitutions in the RBD of omicron, such as Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K, have conferred higher binding afnity to ACE-2.Te furin cleavage site (FCS), at the junction of S1 and S2, plays a key role in the fusion of the virus with the host cell.Omicron contains 3 substitutions (N679K, H655Y, and P681H) close to the furin cleavage site.15 RBD and 3 furin cleavage site substitutions in omicron suggest a major change in infectivity [15].
Before the omicron variant, monoclonal antibody therapy has proven to be highly efective in preventing death; however, it may not be as efective for omicron variants [13].Omicron variants contain mutations within RBD that were previously considered highly conserved and are targets of monoclonal antibodies.Among the 15 RBD substitutions in the omicron variant, the K417N substitution (which is also present in the beta variant) is responsible for the most signifcant disruption to known mAbs [7].Some cases of Q493R mutations following bamlanivimab/etesevimab administration were also reported and are associated with reduced viral clearance and causing fatal outcomes for some patients [13,16].Several omicron sublineages (BA.1, BA.2, and BA.3) shared the same mutation of Q493R, while BA.2 does not have ΔH69/ V70.Omicron lineage BA.4/5 S sequences are identical and closely related to BA.2.Compared to BA.2, BA.4/5 has residues ΔH69/V70, revertant mutant Q493R, and two additional mutations in RBD (L452R and T478K).Tese two additional mutations are considered the main factors for antibody escape [12,17].Based on that, ΔH69/V70 and Q493R mutations are important mutations for omicron to increase its transmissibility and virulence.Terefore, these two mutation sites are used as specifc markers for omicron detection.
Te omicron variant is reported to have mild COVID-19 disease, even though the omicron variant has high infectivity [18].Te severity of the omicron infection is found to be less than that of the delta variant.Te most common symptoms for the omicron variant are coryza, cough, headache, and muscle or limb pain in contrast to the delta variant infection which has common symptoms of smell loss, taste loss, fever, and shortness of breath [19,20].Diarrhea, headache, and shortness of breath appear to be the most important symptoms for the lambda variant [21].Patients infected during the omicron wave were 25% less likely to be admitted to hospital (1.9%) than patients infected during the period of high delta prevalence (2.6%).Patients infected during the omicron wave were also 2.5 times more likely to recover within one week than patients with the delta variant [22].Te previous study found that the delta variant causes long COVID (43%) more common than the omicron variant 2 Advances in Virology (8.2%) [23].Terefore, it is very important to know the variants of SARS-CoV-2.
To handle COVID-19 cases in Indonesia efectively, it is essential to determine the variant type of SARS-CoV-2.Terefore, it is important and necessary to study the validity of SARS-CoV-2 variant determination using the qRT-PCR examination method for SNP.Tis study aims to determine the validity of single-nucleotide polymorphism (SNP) polymerase chain reaction (PCR) variants of omicron SARS-CoV-2 in comparison with the gold standard whole genome sequencing (WGS).

Materials and Methods
Tis is an analytical diagnostic cross-sectional study.Subjects of this study confrmed COVID-19 nasopharyngeal samples were taken from the Laboratory of Kemayoran COVID Emergency Hospital and the West Java Provincial Health Laboratory in the period of April 2022-October 2022.Te workfow diagram for SNP and WGS detection is shown in Figure 1.
2.1.RNA Extraction.RNAs were extracted using the viral PureLink reagent manual method RNA/DNA kits (Invitrogen, Cat.#12280050).Te extraction process uses the manual method of the PURELINK reagent as follows: add 60 mL of 96-100% ethanol to 15 mL of wash bufer and incubate at room temperature.25 μL of Proteinase K was added into a sterile centrifuge tube of 200 μL specimens, positive control or negative control, and 10 μL internal control.200 μL lysis bufer (containing 5.6 μg carrier RNA) was added.Te tube was closed with a tube cover, homogenized with a vortex for 15 seconds, incubated at 56 °C for 15 minutes, and then centrifuged to remove air bubbles.250 μL of 96-100% ethanol was added to the lysate tube for getting 37% ethanol concentration, vortexed for 15 seconds, incubated for 5 minutes at room temperature, and then centrifuged.Te lysate mixed with ethanol was transferred into the spin column and then centrifuged at 6800 × g for 1 minute.Te result of the spin column was placed into a clean wash tube (2 mL); then, 500 μL of wash bufer with ethanol was added to the spin column, turning it at a speed of 6800 × g for 1 minute.Te lysate mixed with ethanol was transferred into the viral spin column and then centrifuged at 6800 × g for 1 minute.Te result was placed into a clean wash tube (2 mL) and then rotated at maximum speed in microcentrifuges for 1 minute.Te viral spin column results were placed into a 1.5 mL tube, and then, 10-15 μL of sterile RNAse-free water was added [24,25].

RT-qPCR Assays.
Te panel mutation reagent used in this study is the TaqMan panel mutation reagent which has been validated by Neopane et al. [26].Te SNP genotyping examination was carried out based on the TaqMan SARS-CoV-2 mutation panel insert kit (Applied Biosystem, Termo Fisher Scientifc).Tis assay required 5 μL Taq Path 1-Step RT-qPCR Master Mix, CG; 0.5 μL TaqMan SARS-CoV-2 Mutation Panel Assay; and 9.5 μL nuclease-free water with a total volume of 15 μL of reagent mix in 96 wells and a 0.2 ml plate.Te reagent mix was mixed with 5 μL of the sample or nuclease-free water, vortexed for 10-30 seconds, and centrifuged for 1-2 minutes at 650 RCF to remove bubbles.Te examination was carried out on QuantStudio 5. Te time taken for the whole process of the qRT-PCR examination method of SNP is one hour and ten minutes [24].
Te TaqMan SNP genotyping assay consists of a sequence-specifc forward and reverse primer that will amplify the sequencing target region.Te reverse primers will transcript the SARS-CoV-2 RNA genome sequence.Each test contains two TaqMan minor groove binder (MGB) probes with a nonfuorescent quencher (NFQ) and a 5′ dye reporter (a VIC dye-labeled probe to detect reference sequences and a FAM dye-labeled probe to detect mutation sequences).
Te samples containing the reference allele will form clusters on the X-axis, and conversely, the samples containing the mutated allele will form clusters on the Y-axis [24].Te plot of genotyping data from TaqMan SARS-CoV-2 mutation panel assays is shown in Figure 2.
Te target gene mutations used in this study were ΔH69/ V70 and Q493R.Omicron sublineages BA.1, BA.2, and BA.3 have the same Q493R mutation, although BA.2 does not have ΔH69/V70.Adversely, omicron sublineages BA.4 and BA.5 have del69-70 but do not have the Q493R mutation [28].Te sample examination was carried out at the Indonesia Research Partnership on Infectious Diseases (INA-RESPOND) Laboratory, Tangerang District Hospital.Te single-nucleotide polymorphism (SNP) criteria for omicron sublineages are shown in Table 1.

Data Analysis. Statistical analysis is carried out with
SPSS version 20, including diagnostic tests (sensitivity, specifcity, positive predictive value, and negative predictive value) and conformity test with Cohen's kappa coefcient test.Te cycle threshold (CT)-value between Omicron sublineages was compared using the Kruskal-Wallis test is shown in Figure 3.

Results and Discussion
A total of 140 nasopharyngeal-positive swab samples were collected from COVID-19 patients who came to Kemayoran COVID Emergency Hospital and the West Java Provincial Health Laboratory in the period of April 2022-October Advances in Virology 2022.Diagnostic criteria for SNPs were made based on gene mutations that occur in each sublineage [9].Te characteristics of the subjects are shown in Table 2.
Most of the subjects were females (59.3%) aged 17-33.Most patients experienced mild symptoms (92.1%), with only two experiencing severe symptoms.It is in line with previous studies that indicate omicron symptoms are less severe than delta ones.Moreover, the provision of COVID-19 vaccination makes the symptoms of COVID-19 milder [19].Te WGS result concluded that most of the samples are omicron BA.4/BA.5 (85%), which is in line with     Cohen's kappa coefcient test to analyze the conformity between the results of SNP and WGS showed high conformity (p value <0.001; kappa coefcient 0.948), as shown in Table 3. Tis result follows a previous study that concluded SNP to have high conformity to WGS and may be useful as an omicron marker, even though validation is required for the given setting [30].
Te cycle threshold (CT) value between omicron sublineages was then compared using the Kruskal-Wallis test (Table 5).Te median CT value for omicron BA.1 is the lowest (17.7;IQR 11.3-25.0),followed by omicron BA.2 (21.1;IQR 11.7-26.7),and omicron BA.4/5 the highest (24.1;IQR 19.1-27.6).Te CT values between the three sublineages show a signifcant diference (p value <0.05) (Figure 1).Mutation Q493R in omicron BA.1 and BA.2 was associated with a rise in SARS-CoV-2 viral load in nasopharyngeal samples; this causes the CT values in BA.1 and BA.2 to be lower than those in other variants.In some cases, the presence of the Q493R mutation was associated with a relapse of COVID-19 with distress respiratory syndrome [13].Meanwhile, the Q493R reversion mutation in omicron BA.4/BA.5 allows it to regain binding ftness and may even lead to a slightly higher afnity of BA.4/ BA.5 for ACE-2 compared to other omicron subvariants.Terefore, symptoms caused by omicron BA.4/BA.5 can be seen in a lower viral load (higher CT-values) than in omicron BA.1 or BA.2 [32].
Te limitation of this study is the small number of samples available, especially for omicron sublineages BA.1 and BA.2, which afects the validity of SNP.Smaller samples than necessary would have insufcient statistical power to Advances in Virology answer the primary research question, and a statistically nonsignifcant result could merely be because of inadequate sample size.Te results of this study may only apply to the population under study; they cannot be generalized to other populations, especially to the omicron sublineages BA.1 and BA.2 [33].Tis study can only detect and diferentiate between omicron BA.1, BA.2, and BA.4/5.Te study could not distinguish BA.4 from BA.5 and could not detect other omicron sublineages.Terefore, additional specifc SNP mutation targets are required to detect and diferentiate other omicron sublineages.

Conclusions
Te use of the SNP method to determine omicron variants has produced a good validity value that conforms to WGS, which is considered the gold standard.Taking into account the efciency of time and facility requirements, the SNP method can be used as an alternative assay to detect omicron variants.

Suggestions.
A further study with a larger number of samples of omicron, especially sublineages BA.1 and BA.2, is recommended to increase statistical power.It is important and necessary to determine specifc mutation targets for SNP to diferentiate sublineages and severity of diferent omicron variants in accordance with the ongoing situation of the pandemic.
Sequencing.Presence of signature mutations was confrmed by whole genome sequences examined by the Health Development Policy Agency of the Ministry of Health Laboratory from the Laboratory of Kemayoran COVID Emergency Hospital and the West Java Provincial Health Laboratory PCR-positive samples.

Table 2 :
Characteristics of the subjects.

Table 3 :
Conformity of SNP and WGS results.

Table 4 :
Diagnostic values of SNP towards WGS.

Table 5 :
Te diference between CT values and WGS results.